1. Field of the Invention
The present invention relates to immunoassays, and, more particularly, to improved microscale diffusion immunoassays utilizing multivalent reactants.
2. Description of the Related Art
The immunoassay is the workhorse of analytical biochemistry. It allows the unique binding abilities of antibodies to be widely used in selective and sensitive measurement of small and large molecular analytes in complex samples. The driving force behind developing new immunological assays is the constant need for simpler, more rapid, and less expensive ways to analyze the components of complex sample mixtures. Current uses of immunoassays include therapeutic drug monitoring, screening for disease or infection with molecular markers, screening for toxic substances and illicit drugs, and monitoring for environmental contaminants.
Flow injection immunoassays have taken advantage of specific flow conditions (see U. de Alwis and G. S. Wilson, Anal. Chem. 59, 2786-9 (1987)), but also use high Reynolds number effects for mixing. Micro-fabricated capillary electrophoresis devices, which are truly microfluidic, have been used for rapidly separating very small volumes of immunoreagents following binding reactions (see N. Chiem and D. J. Harrison, Anal. Chem. 69,373-8 (1997)). However, one of the unique features of microfluidic devices that has yet to be fully exploited for immunoassay development is the presence of laminar flow under low Reynolds number conditions. Laminar flow allows for quantitative diffusional transport between adjacent flowing streams, while retaining the relative positions of essentially non-diffusing components such as cells and larger microspheres. While these conditions are impediments to the application of some macro-scale techniques, they allow for the creation of new types of analyses that are uniquely well suited to microfluidic systems, such as the H-Filter for extraction of solutes (see J. P. Brody, P. Yager, R. E. Goldstein and R. H. Austin, Biophysical Journal 71(6), 3430-3441 (1996); U.S. Pat. No. 5,932,100; and J. P. Brody and P. Yager, Sensors and Actuators A (Physical) A58(1), 13-18 (1997)), the V-Groove device for low-volume flow cytometry (see U.S. Pat. No. 5,726,751), the T-Sensor for detection of diffusable analytes (see A. E. Kamholz, B. H. Weigl, B. A. Finlayson and P. Yager, Anal. Chem. 71(23), 5340-5347 (1999); U.S. Pat. No. 5,716,852; U.S. Pat. No. 5,972,710; B. H. Weigl and P. Yager, Science 283, 346-347 (1999); R. B. Darling, J. Kriebel, K. J. Mayes, B. H. Weigl and P. Yager, Integration of microelectrodes with etched microchannels for in-stream electrochemical analysis, μTAS '98, Banff, Canada (1998); B. H. Weigl and P. Yager, Sensors and Actuators B (Chemical) B39 (1-3), 452-457 (1996); B. H. Weigl, M. A. Holl, D. Schutte, J. P. Brody and P. Yager, Anal. Methods & Instr. 174-184 (1996); and B. H. Weigl et al., Simultaneous self-referencing analyte determination in complex sample solutions using microfabricated flow structures (T-Sensors), μTAS '98, Banff, Canada (1998)); and others as described in U.S. Pat. No. 5,922,210; U.S. Pat. No. 5,747,349; U.S. Pat. No. 5,748,827; U.S. Pat. No. 5,726,404; U.S. Pat. No. 5,971,158; U.S. Pat. No. 5,974,867; U.S. Pat. No. 5,948,684; WO 98/43066, published Oct. 1, 1998; U.S. patent application Ser. No. 08/938,584, filed Sep. 26, 1997; WO 99/17100, published Apr. 8, 1999; WO 99/17119, published Apr. 8, 1999; U.S. patent application Ser. No. 09/196,473, filed Nov. 19, 1998; U.S. patent application Ser. No. 09/169,533, filed Oct. 9, 1998; WO 99/60397, published Nov. 25, 1999; U.S. patent application Ser. No. 09/404,454, filed Sep. 22, 1999; and U.S. patent application Ser. No. 09/464,379, filed Dec. 15, 1999.
A number of microscale diffusion immunoassays (DIAs) were disclosed in U.S. Pat. No. 6,541,213 (“the '213 patent”), which patent is incorporated herein by reference in its entirety. As described therein, such DIAs may be utilized to determine the presence and concentration of an analyte in an analyte fluid by: flowing the analyte fluid, comprising analyte particles, and a diffusion fluid, comprising binding particles (such as antibodies), in adjacent laminar flow in a microfluidic laminar flow channel; allowing the analyte particles to diffuse into the diffusion fluid and bind with the binding particles to form analyte/binding particle complexes; and detecting the presence of said analyte particles, both complexed and uncomplexed. As further noted in the '213 patent, such a DIA relies on the change in the transport properties of the analyte when it becomes complexed with a binding particle (i.e., the diffusion profile of the analyte is modified by the binding particle). In particular, it is noted that the analyte/binding particle complexes diffuse more slowly than the analyte in its unbound state, and, therefore, a population of such complexes will accumulate near the diffusion interface between the two fluid streams. By monitoring the diffusion profile of the analyte (both bound in a complex and unbound) for any changes, the binding event can be detected without the need for separation of the analyte/binding particle complexes from the original populations of analyte and binding particles and without the requirement that the properties of the analyte particles change with binding.
However, in order for changes in the diffusion profile of the analyte to be detectable in the foregoing DIA, the analyte/binding particle complexes must have significantly different diffusion properties than the unbound analyte particles. For example, as noted in the '213 patent, the ratio of diffusivity of the complexes to the diffusivity of the unbound analyte particles should be greater than about two in order for the DIA response to be resolvable from the experimental noise levels typical for the DIA. This creates a limit on the size of the analyte particles depending on the size of the binding particles. For example, if the binding particles are antibodies (MW ˜150 kD), then the analyte particles cannot exceed ˜45 kD. To detect a larger analyte, the binding particles must be correspondingly larger, such as an antibody immobilized on the surface of a microsphere (see K. R. Hawkins, A. Hatch, H. Chang and P. Yager, “Diffusion Immunoasay of Protein Analytes,” 2nd Annual International Conference IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine and Biology, May 2-4, 2002, Madison, Wis., USA, 2002).
Accordingly, although there have been advances in the field, there remains a need in the art for new and improved microscale diffusion immunoassays, in particular, DIAs having increased response sensitivity, which are not subject to the above limitations. The present invention addresses these needs and provides further related advantages.